The present invention relates to a novel monooxygenase having, in the same molecule, a monooxygenase activity and a reducing power supplying ability from NADPH required for said monooxygenase activity; genes which code for said monooxygenase; yeast expression plasmids containing said genes; and yeast strains transformed with said expression plasmids.
More particularly, it relates to monooxygenase having, in the same molecule, a monooxygenase activity derived from cytochrome P-450 (hereinafter referred to as "P-450") and reducing power supplying ability from NADPH derived from NADPH-cytochrome P-450 reductase (hereinafter referred to as "reductase"); chimeric fused enzyme genes which code for said monooxygenase; yeast expression plasmids containing said genes; and yeast strains transformed with said expression plasmids and to a process for producing said monooxygenase by cultivating said transformed yeast strains.
P-450 is a heme protein existing widely in biological fields from microorganisms to mammals and catalyzes monooxygenase activity toward a wide variety of lipophilic compounds as substrates. Such a wide variety of substrate specificity exhibited by P-450 are attributable to molecular diversity of P-450. That is, there are many molecular forms of P-450 whose substrate specificity is wide and overlapping each other. Many of them are common in electron transfer routes. In case of liver microsome, only one form of reductase containing flavin adenin mononucleotide and flavin mononucleotide as coenzymes mainly supplies electrons from NADPH to substrate-bounded P-450. Therefore, P-450 exhibits monooxygenase activity only when it binds a substrate and couples with reductase.
We have already succeeded in production of enzyme proteins which show monooxygenase activity by isolating P-450MC and reductase genes present in rat liver and expressing these genes in yeasts as their hosts P-450 MC is a gene coding for rat liver cytochrome P-450 inducible by 3-methylcholanthrene (MC refers to 3-methylcholanthrene) [Oeda et al., DNA Vol. 4 No. 3 p203-210 (1985); Murakami et al., DNA Vol. 5 No. 1 p. 1-10 (1986)]. P-450 MC synthesized in yeasts constituted electron-transport chains in yeast microsome by coupling with yeast reductase and exhibited monooxygenase activity inherent to rat P-450MC. The P-450MC-producing yeast strains were able to convert acetanilide to acetaminophene useful as a medicine.
Therefore, P-450MC-producing yeast strains or P-450MC obtained from transformed yeasts can be applied to oxidative reaction process of useful substances and further to oxidative removal of harmful substances from industrial waste.
We have made researches in an attempt to enhance monooxygenase activity of P-450MC and produced yeast strain which produces P-450MC and reductase simultaneously [Murakami et al, DNA Vol. 5 No. 1 p. 1-10 (1986)].